The production of optically active organic compounds, e.g. alcohols and amino acids, by a biocatalytic route is increasingly gaining importance. The coupled use of two dehydrogenases with cofactor regeneration has emerged as a route for the large-scale industrial synthesis of these compounds (DE19753350).

In situ regeneration of NADH with the NAD-dependent formate dehydrogenase (“FDH”) in the reductive amination of trimethyl pyruvate to give L-tert-leucine (Bommarius et al. Tetrahedron Asymmetry 1995, 6, 2851-2888).
In addition to their catalytic property and efficiency, the biocatalysts efficiently employed in an aqueous medium furthermore have the advantage that in contrast to a large number of synthetic metal-containing catalysts, the use of metal-containing starting substances, in particular those which contain heavy metals and are therefore toxic, can be dispensed with. The use of expensive and furthermore hazardous reducing agents, such as, for example, borane, in the case of asymmetric reduction can also be dispensed with.
Nevertheless, difficulties occur in the reaction of substrates which are poorly water-soluble. This affects in particular the preparation of alcohols from hydrophobic carbonyl compounds, in which the substrate solubility is often below 10 mM. Similar difficulties exist in the case of poorly water-soluble products. A solution which is conceivable in principle would be to carry out the biocatalytic reduction in a polar organic solvent or a resulting homogeneous aqueous solution thereof. In this case, both the enzymes and the substrate and, where appropriate, the product should be water-soluble. A general disadvantage of a direct presence of an organic solvent, however, is the considerable reduction which generally occurs in the enzyme activity under these conditions (see e.g. Anderson et al., Biotechnol. Bioeng. 1998, 57, 79-86). In particular, FDH as the only formate dehydrogenase employed hitherto on an industrial scale and accessible in commercial amounts unfortunately has a high sensitivity towards organic solvents. This also manifests itself in the comparison examples 1 using DMSO, sulfolane, MTBE, acetone, isopropanol and ethanol as the organic solvent component in added amounts of in each case 10% (see FIG. 1).
Various set-ups are known to solve this problem relating to stabilization of the formate dehydrogenase from Candida boidinii in the presence of organic solvents, e.g. carrying out reactions by the additional use of surfactants as surface-active substances. Disadvantages here, however, are the rate of reaction, which is reduced by about a factor of 40 (!), and the inhibition of formate dehydrogenase which occurs (B. Orlich et al., Biotechnol. Bioeng. 1999, 65, 357-362). The authors furthermore note that because of the low stability of the alcohol dehydrogenase employed, a reduction process under these conditions of a microemulsion is not economical. In addition, there is a further problem in the working up, in which the resulting product must be separated from the surfactant, which has often proved to be not a trivial matter.
A possibility in principle also consists of carrying out enzymatic reactions or oxidations in a two-phase system. Here however—analogously to the abovementioned destabilizing effects in the presence of organic water-soluble solvents—only a particular class of organic solvents, namely those with a very hydrophobic character, such as, for example, heptane and hexane, has proved to be suitable. On the other hand, stability studies with other nonpolar solvents, such as toluene, but above all with typical solvents such as MTBE and ethyl acetate, showed a drastic decrease in the activity of the formate dehydrogenase from Candida boidinii even in a very short service life (H. Groger et al., Org. Lett. 2003, 5, 173-176). In the presence of heptane and hexane, in contrast, the reaction can indeed be carried out, but the solubility of the ketone substrates in these solvents is often limited.
A further possibility in principle for carrying out biocatalytic reactions consists of the use of immobilized enzymes in the organic solvent or the use of enzymes in a homogeneous solution comprising water and a water-miscible organic solvent. However, these techniques in which direct contact occurs between the organic solvent and enzyme are limited to a few enzyme classes, in particular hydrolases. It is thus noted in DE4436149 that the “direct presence of organic solvents (water-miscible or water-immiscible) is tolerated by only a few enzymes which belong to the class of hydrolases”. A few further examples from other enzyme classes have indeed since become known (thus, inter alia, oxynitrilases), but the statement made in DE4436149 is still applicable to the majority of enzymes. An efficient immobilization of the FDH from Candida boidinii is thus not known. Rather, for example, it is known with the Eupergit method, as a standard tool of industrial immobilization, that the residual activity of this FDH after immobilization is <20%, which is too low for an industrial utilization. Furthermore, the immobilization itself is associated with additional costs due to the immobilization step and the immobilization materials.
Industrially, processes have therefore been developed which avoid the presence of organic solvents because of the risk of deactivation or denaturing of the enzymes. DE4436149 thus describes a process in which the product is extracted from the reaction solution into an organic solvent through a membrane, in particular a hydrophobic membrane, which is permeable to the product. Compared with a standard process in a stirred tank reactor, however, this process requires significantly more technical outlay, especially since the organic membranes required are also an additional cost factor. Furthermore, this method is suitable only for continuous processes. In addition, the disadvantage in principle of carrying out the reaction at low substrate concentrations also cannot be avoided with this method. Accordingly, the substrate concentrations are below the solubility limit, which for most ketones is 10 mM or considerably lower. However, substrate concentrations of 100 mM or above would be desirable for an industrial reaction.
Summarizing, it can be said that thus no process which helps to bypass the abovementioned disadvantages is known.